Rolling and arrest of platelets on surfaces with various extracellular matrix (ECM) proteins under shear stress is central to hemostasis. Similarly, rolling and arrest of leukocytes (neutrophils and monocytes) on surfaces with various adhesion molecules plays an important role in inflammation and atherosclerosis. The two processes can be studied ex vivo by perfusion of blood over substrates with various coatings and monitoring the dynamics of rolling and arrest of platelets or leukocytes, as appropriate. An important component of both the dynamic platelet adhesion assay and the leukocyte rolling assays is the coating of the glass substrate, known as a flow chamber, with adhesion molecules: VWF, fibrinogen, and collagen for platelets, and selectins, chemokines, ICAM-1 and CAM-1 for leukocytes. Flow chambers have been in use since the early twentieth century for various tissue-culture applications, and were first used to study rolling and arrest in the 1980's.
Another application of flow chambers is the study of shear stress responses of endothelial cells. Endothelial cells form a monolayer on the interior surface of blood vessels and sense shear stress generated by blood flow. Endothelial cells respond to shear stress with rearrangements, vascular remodeling, and alterations of vascular tone and vascular permeability. Shear stress can modulate many functions of endothelial cells including gene expression, cell adhesion, proliferation, differentiation, migration, and cytoskeletal alignment relative to the direction of flow.
Perfusion chambers for experiments with live cells are among the most straightforward and versatile microfluidic devices. In addition to substantial reduction of the amounts of media and cells required for an experiment as compared to traditional flow chambers, microfluidic perfusion chambers offer numerous new capabilities. Those include the variation of composition of the perfusion medium across the microfluidic chip and in time, the capture of flowing cells using microfabricated weirs or posts, and generation of substrate coatings with customized micro-patterns. Microfluidic perfusion devices that are made of a cast PDMS (polydimethylsiloxane) chip sealed with a microscope cover glass also offer the advantages of compatibility with the standard high-resolution microscope objective lenses as well as low cost and disposability. Microfabrication makes it easy to produce tapered perfusion chambers, which generate varying shear stresses at the substrate.
A significant drawback, however, is that the loading of cells into such microfluidic devices can be a delicate task. If the cell stock is small, cells can be sensitive to hydrodynamic stresses, or a particular cell density on the substrate may need to be reached. The pre-assembled microfluidic devices that are commonly used for rolling and arrest studies are coated by perfusing the microchannels with solutions of the adhesion molecules and incubating the solutions in the microchannels. This coating procedure can make it difficult to measure and control the site density of the adhesion molecules. One solution to address this problem is to use excessive concentrations of adhesion molecules in an attempt to achieve some unknown saturated site density. The problem with this method is that, along with the flow shear stress, the site density is an essential experimental parameter. This approach also requires substantially more reagents and does not allow creation of substrate regions with different coatings on them. The ability to control the site density of the adhesion molecules should make it easier to emulate in vivo conditions, and potentially more importantly, to improve the repeatability of the assays and to provide new opportunities for detection of adhesion (and rolling) phenotypes of certain mutations and modifications of integrins and related molecules.
A different approach to address the site density problem is to incubate the glass substrate under a layer of solution with a known concentration of adhesion molecules. While this would be expected to improve control and reproducibility of the site density, a major obstacle is that microfluidic chips made of PDMS are normally sealed against dry cover glasses using either oxygen plasma or relatively high temperatures, both of which destroy biomolecular coating on the glass surface. Thus, the use of cover glasses with pre-coated surfaces requires finding a way to seal PDMS chips against wet cover glasses.
Two main techniques have been proposed to seal PDMS microchannel chips against wet substrates with cell cultures: mechanical clamping and vacuum suction, both of which introduce their own problems. The application of mechanical clamps usually involves poorly characterized and not completely reproducible mechanical stresses that may cause substantial deformations of microchannels, which may vary across the device. Sealing by vacuum suction is achieved by surrounding the “wet” microchannel array, containing media and cells, with a secondary network of “dry” microchannels connected to a source of vacuum. This technique has been used for short term experiments with endothelial cells and for long-term stem cell cultures. However, the application of vacuum could produce slow changes in the gas content of the wet channel medium that may be difficult to detect and quantify. Thus, both mechanical clamping and vacuum suction introduce variables into the experiment or assay that negatively impact accuracy and reproducibility.
Accordingly, the need remains for a device and method for sealing microchannel chips against wet substrates without damaging the cells or introducing conditions that can affect accuracy and reproducibility of the experiments. Further, a need remains for a method for perfusing microfluidic chips that provides control of site density for improved repeatability and expanded experimental applications.